The present invention relates to a novel process for the purification of an outer membrane protein of Bordetella pertussis, having a molecular weight of approximately 69,000 Daltons, formerly called the 69 kDa protein and now called pertactin, and obtained from the fermentation broth and cellular extracts of the said organism. The protein obtained by the process is to be used in a xe2x80x9ccomponentxe2x80x9d vaccine to protect against the disease of whooping cough.
The disease of whooping cough or pertussis is a result of infection by Bordetella pertussis, and is a serious and debilitating human disease particularly in young children. For the last fifty years the disease has been controlled through large-scale immunization programmes. The current licensed vaccine in North America is a xe2x80x9cwhole cellxe2x80x9d vaccine prepared by growing the organism in fermentors and then treating the resulting B. pertussis cells with chemical agents, such as formaldehyde, to kill the organism and inactivate toxic proteins. The cells are resuspended and then used directly or in combination with other antigens. This vaccine, although highly efficacious, has been associated with clinical symptoms that include fever, local reactions, high-pitched crying and convulsions. Despite the fact that there is no proven relation between these symptoms and the vaccine, there has been decreased public acceptance of this vaccine and in a number of countries, e.g. Japan, Sweden and the U.K., decreased immunization has led to outbreaks of the disease. The need for a more defined vaccine has been recognized and considerable effort has been directed by several manufacturers and researchers towards the development of an efficacious pertussis vaccine that consists of a small number of highly purified proteins. This vaccine has been termed a component vaccine.
This search has been hampered by a lack of information on the mechanism of pathogenesis of B. pertussis. Many virulence associated factors, such as pertussis toxin (PT), also known as lymphocytosis promoting factor (LPF), filamentous haemagglutinin (FHA), adenylate cyclase, lipopolysaccharide, agglutinogens and other outer membrane proteins have been suggested for inclusion in an xe2x80x9cacellularxe2x80x9d vaccine, which is less defined than the component vaccine. Much of the work on acellular vaccines has concentrated on a PT-based vaccine. Results of a recent clinical trial have indicated that a vaccine consisting entirely of PT-toxoid only partially protected children from the infection. A PT/FHA combination showed slightly higher efficacy but this was still lower than that obtained for the whole-cell vaccine.
One potential protective antigen is an outer membrane protein, with a molecular weight of approximately 69,000 daltons (pertactin) found on all virulent strains of B. pertussis. This protein is produced in relatively large amounts during the culture of the organism and can be purified from either the fermentation broth or from cell extracts. The present invention provides a novel method of effecting such purification.
The potential importance of this outer membrane protein for inclusion in a human vaccine against whooping cough was suggested from attempts to prepare a vaccine to protect pigs against B. bronchiseptica infection. Cell-surface extracts of B. bronchiseptica were used to immunize sows. Levels of antibody to a cell surface antigen with a molecular weight of 68,000 daltons correlated with protection of newborn piglets against infection. Similar antigens, with similar molecular weights, were detected in B. pertussis (approximately 69,000 daltons) and in B. parapertussis (approximately 70,000 daltons). Immunization with the protein obtained from B. pertussis protected mice against intracerebral challenges with live organisms and antibodies to the protein conferred passive protection to mice in this test. Both active and passive protection of mice in an aerosol challenge model have also been described.
The published procedures for purification of pertactin do not allow for the large-scale production of a highly purified, non-pyrogenic and stable antigen. One reported method (Canadian Patent No. 1,253,073) involves acid-glycine extraction of the cells, anion-exchange chromatography and preparative iso-electric focussing. However, the pertactin obtained has been reported to degrade into smaller fragments, to be sensitive to low pH and to have adenylate cyclase activity. For these reasons, this extraction procedure is considered undesirable for large-scale production. In addition, iso-electric focussing is not amenable to large-scale production. A second procedure (U.S. Pat. No. 5,101,014) involved the extraction of the outer membrane protein from the cells of an afimbriated strain of B. pertussis. The protein was purified by a combination of DEAE-Sepharose and Affigel-blue chromatographies. The potential of leaching the blue dye into the product would be a possible safety concern. Neither method addresses the purification of pertactin directly from fermentation broths.
In one embodiment of the present invention, pertactin is obtained in large quantities from fermentor broth, which is the preferred source, and in a purified form, by using the method described below. The protein can be included in a product to be used for the widespread vaccination of children against whooping cough.
After growing the organism in a fermentor, the cells are removed by centrifugation and filtration and the supernatant reduced in volume and sterilised. The broth is diluted to a low ionic strength and, after removal of other antigens, the pertactin is isolated by chromatography on various substrates and further purified by ultrafiltration. The protein can also be isolated from the cells after extraction with urea, centrifugation and further processing to give a solution that can be treated as described above.
Accordingly, in one aspect, the present invention provides a method for the production of pertactin, which comprises providing an impure aqueous solution of pertactin substantially free from other Bordetella antigens, purifying pertactin in said aqueous solution by passing said aqueous solution sequentially in contact with hydroxyapatite and an ion-exchange medium, and subjecting the resulting purified solution to ultrafiltration.
The process described in this invention allows for the purification of several protein antigens for possible inclusion in a component pertussis vaccine from a single fermentation of B. pertussis. 
In the present invention, B. pertussis is grown in a fermentor under controlled conditions. Carbon sources and growth factors are supplemented either continuously or in batches at various intervals during the fermentation until the pertussis proteins (PT, FHA and pertactin) are at the desired levels as determined by a specific enzyme-linked immunosorbent assay (ELISA) for each antigen. The fermentor broth is harvested, the majority of the cells removed by centrifugation and the broth sterilised by microfiltration, preferably using known membrane filters of about 0.2 micron pore size. The broth is concentrated, say 10-fold, by membrane ultrafiltration and used for the purification of pertussis toxin and FHA (see published European Patent Application No. 0,336,736; U.S. Pat. No. 4,997,915, the disclosure of which is incorporated herein by reference). The cells are the source of material for the purification of the agglutinogens. Pertactin can be purified from both the broth or cells. The former is preferred as the majority of the protein is found in the broth.
The first stage in the purification of the pertactin requires dilution of the broth to a low ionic strength and chromatography on Perlite or other suitable solid particulate adsorbent material to remove the PT and FHA antigens, as more fully described in the aforementioned published European Patent Application No. 0,336,736, U.S. Pat. No. 4,997,915). The PT and FHA antigens can be removed from the adsorbent material by treatment with an aqueous medium of high ionic strength for use in a component pertussis vaccine.
As used herein, the term xe2x80x9clow ionic strengthxe2x80x9d refers to an aqueous medium having a conductivity of about 11 mS/cm or less, preferably about 4 mS/cm or less. The unit of measurement mS/cm is millisiemen per centimeter. A Siemen (S) is a unit of conductivity and is the equivalent of the inverse of resistance (ohm) and is sometimes designated mho. The term xe2x80x9chigh ionic strengthxe2x80x9d as used herein refers to an aqueous medium having a conductivity of greater than about 11 mS/cm and preferably at least about 50 mS/cm.
The remaining mixture then is concentrated by membrane filtration, subjected to ammonium sulphate precipitation and the resulting pellet dissolved in low ionic strength buffer, such as Tris-.HCl, at a pH of 6.0 to 8.5 to yield a solution with a final conductivity of generally less than about 4 mS/cm, typically approximately 3.4 mS/cm. The solution is chromatographed sequentially on hydroxyapatite, and an ion-exchange medium, such as Q-Sepharose(copyright). Pertactin elutes in the unbound fraction of both columns under the specified buffer conductivity. However, if the conductivity is lower than 1.5 mS/cm for hydroxyapatite or 2.8 mS/cm for Q-Sepharose(copyright), pertactin binds to both columns and can be eluted with a buffer having conductivities 1.5 mS/cm or greater for hydroxyapatite and 2.8 mS/cm for or greater Q-Sepharose(copyright).
The pertactin is further purified by ultrafiltration through about 100 to 300 kDa Nominal Molecular Weight Limit (NMWL) membranes where it is collected in the filtrate, concentrated using membranes with a NMWL of about 30 kDa or less and sterile filtered for use in combination with other pertussis antigens in a vaccine.
In an alternative procedure, pertactin along with FHA and PT are precipitated from fermentor broth, by addition of ammonium sulphate. The precipitate is removed from the residual broth and redissolved to provide an aqueous solution suitable for processing as described above first to remove the PT and FHA and then to purify the pertactin.
Pertactin also can be purified from B. pertussis cells. The cells are extracted in a solution containing a high concentration (for example, 4 M) of urea for, say 1.5 hr. at room temperature. Cell debris is removed by centrifugation and the supernatant, which contains the protein, is subjected to ultra-filtration using 100 to 1000 kDa NMWL membranes. High molecular weight proteins, such as the agglutinogens, are retained while the majority of the pertactin is filtered through. The filtrate is concentrated, diafiltered using a 30 kDa or less NMWL membrane, and then precipitated by ammonium sulphate and centrifuged to give a pellet for processing as described above.
Pertactin, although reportedly susceptible to proteolytic cleavage, is very stable when purified by the method of the invention. SDS-PAGE analysis indicates that the purified pertactin is homogenous and essentially intact with traces of degradation products of molecular weights between 30 and 40 kDa. No evidence for further degradation or change in immunogenicity was found after several months of storage at 2 to 8xc2x0 C. or at higher temperatures (24xc2x0 C., 37xc2x0 C.). In contrast to a previous publication (Canadian Patent No. 1,253,073), the finding that the pertactin prepared by this method does not show any detectable adenylate cyclase activity and has enhanced stability makes it an ideal candidate for inclusion into a component vaccine for protection against B. pertussis.